AGEs increase lipocalin 2 expression

Background and Objectives: Diabetes mellitus (DM), a risk factor of periodontal diseases, exacerbates the pathological condition of periodontitis. A major factor for DM complications is advanced glycation end-products (AGEs) that accumulate in periodontal tissues and cause inflammatory events. Lipocalin 2 (LCN2) is an antimicrobial peptide and inflammation-related factor, and LCN2 levels increase in DM. In the present study, the effects of AGEs and lipopolysaccharide of Porphyromonas gingivalis (P.g-LPS) on LCN2 expression in human oral epithelial cells (TR146 cells) and the role of secreted LCN2 in periodontitis with DM were investigated. Material and Methods: TR146 cells were cultured with AGEs (AGE2) and control BSA and cell viability was estimated, or with P.g-LPS. Conditioned medium and cell lysates were prepared from cultures of epithelial cells and used for western blotting and ELISA to analyze LCN2, RAGE, IL-6, MAPK and NF-κB. RNA was isolated from AGE-treated TR146 cells and differentiated HL-60 (D-HL-60) cells and used for quantitative real-time PCR to examine the expression of LCN2 and interleukin-6 (IL-6) mRNAs. RAGE- and LCN2-siRNAs (siRAGE, siLCN2) were transfected into epithelial cells, and AGE-induced LCN2 expression was investigated. D-HL-60 cells were co-cultured with TR146 cells that were transfected with siLCN2 and treated with AGEs, IL-6 mRNA expression in D-HL-60 cells and cell migration were investigated. Results: AGEs increased the expression levels of LCN2 and IL-6 in oral epithelial cells. siRAGE and a neutralizing antibody for RAGE inhibited AGE-induced LCN2 expression. AGEs stimulated the phosphorylation of ERK, p38 and NF-kB in epithelial cells, and their inhibitors suppressed AGE-induced LCN2 expression. In contrast, P.g-LPS did not show a significant increase on LCN2 level in TR146 cells that expressed toll-like receptor 2. In co-culture experiments, AGE-induced LCN2 inhibited IL-6 mRNA expression in D-HL-60 cells, and LCN2 knockdown in epithelial cells suppressed HL-60 cell migration. Conclusion: These results suggested that AGEs increase LCN2 expression via RAGE, MAPK, and NF-κB signaling pathways in oral epithelial cells, and secreted LCN2 may influence the pathological condition of periodontitis with DM

Gan-Lu-Yin (Kanroin), Traditional Chinese Herbal Extracts, Reduces Osteoclast Differentiation In Vitro and Prevents Alveolar Bone Resorption in Rat Experimental Periodontitis

Gan-Lu-Yin (GLY), a traditional Chinese herbal medicine, shows therapeutic effects on periodontitis, but that mechanism is not well known. This study aims to clarify the precise mechanism by investigating the inhibitory effects of GLY extracts on osteoclastogenesis in vitro and on bone resorption in periodontitis in vivo. RAW264.7 cells are cultured with soluble receptor activator of nuclear factor-kappa B (sRANKL) and GLY extracts (0.01–1.0 mg/mL), and stained for tartrate-resistant acid phosphatase (TRAP) to evaluate osteoclast differentiation. Experimental periodontitis is induced by placing a nylon ligature around the second maxillary molar in rats, and rats are administered GLY extracts (60 mg/kg) daily for 20 days. Their maxillae are collected on day 4 and 20, and the levels of alveolar bone resorption and osteoclast differentiation are estimated using micro-computed tomography (CT) and histological analysis, respectively. In RAW264.7 cells, GLY extracts significantly inhibit sRANKL-induced osteoclast differentiation at a concentration of more than 0.05 mg/mL. In experimental periodontitis, administering GLY extracts significantly decreases the number of TRAP-positive osteoclasts in the alveolar bone on day 4, and significantly inhibits the ligature-induced bone resorption on day 20. These results show that GLY extracts suppress bone resorption by inhibiting osteoclast differentiation in experimental periodontitis, suggesting that GLY extracts are potentially useful for oral care in periodontitis

Wall-yielding properties of cell walls from elongating cucumber hypocotyls in relation to the action of expansin

The wall-yielding properties of cell walls were examined using frozen-thawed and pressed segments (FTPs) obtained from the elongation zones of cucumber hypocotyls with a newly developed programmable creep meter. The rate of wall extension characteristically changed depending on both tension and pH. By treatment of the FTPs with acid, the yield tension (y) was shifted downward and the extensibility (f) was increased. However, the downward shift of y was greatly suppressed and the increase in f was partly inhibited in boiled FTPs. The boiled FTPs reconstituted with expansin fully recovered the acid-induced downward y shift as well as the increase in f. Even under the tension below y, wall extension took place pH dependently. Such extension was markedly slower (low-rate extension) than that under the tension above y (high-rate extension). At a higher concentration (8 M), urea markedly inhibited the creep ascribable to the inhibition of the acid-induced downward y shift and increase in f. Moderate concentrations (2 M) of urea promoted wall creep pH dependently. The promotion was equivalent to a 0.5 decrease in pH. The promotion of creep by 2 M urea was observed in boiled FTPs reconstituted with expansin but not in boiled FTPs. These findings indicated that the acidfacilitated creep was controlled by y as well as f in cucumber cell walls. However, y and f might be inseparable and mutually related parameters because the curve of the stress extension rate (SER) showed a gradual change from the low-rate extension to the high-rate extension. Expansin played a role in pH-dependent regulation of both y and f. The physiological meaning of the pH-dependent regulation of wall creep under different creep tensions is also discussed with reference to a performance chart obtained from the SER curves

S100A9は、骨細胞様細胞においてMAPKsおよびSTAT3シグナル伝達経路を介してIL-6およびRANKLの発現を増加させる

Objective: Calprotectin is hetero-complex of S100A8 and S100A9 and mainly secreted from neutrophils, monocytes and chondrocytes in inflammatory condition. Calprotectin binds to RAGE and TLR4, and induces the expression of pro-inflammatory chemokines and cytokines in various cells. Periodontitis is chronic inflammatory disease to lead gingival inflammation and alveolar bone resorption. Calprotectin levels in gingival crevicular fluid of periodontitis patients are higher than healthy patients. In the present study, the effects of S100A8 and S100A9 on the expressions of pro-inflammatory cytokines and bone metabolism related factor in mouse osteocyte like cells (MLO-Y4-A2) were investigated. Design: MLO-Y4-A2 cells were treated with S100A8 and S100A9, and the expressions of RAGE, TLR4, RANKL and several inflammatory cytokines were analyzed by PCR and Western blotting or ELISA methods. To investigate the intracellular signaling pathways, phosphorylation of MAPK and STAT3 was determined by Western blotting, and chemical specific inhibitors and siRNAs were used. Results: Expressions of IL-6 and RANKL were increased by treatment with S100A9 but not S100A8. However, both S100A8 and S100A9 did not changed expression of IL-1β, IL-8 and TNF-α. Although RAGE and TLR4 expressions were not up-regulated by S100A9 treatment, transfection of siRNA for RAGE and TLR4 significantly decreased IL-6 and RANKL expressions. In addition, S100A9 activated p38, ERK and STAT3 signaling pathways, and inhibitors for these factors significantly decreased S100A9 induced IL-6 and RANKL expressions. Conclusions: These results indicated that S100A9 induces IL-6 and RANKL production via engagement with RAGE and TLR4 signalings in osteocytes and suggested that S100A9 may play important roles in the periodontal alveolar bone destruction

ショウガオールはヒト歯肉線維芽細胞において酸化ストレス反応の調節を介してAGEs誘導性のIL-6およびICAM-1産生を抑制する

Advanced glycation end-products (AGEs) cause diabetes mellitus (DM) complications and accumulate more highly in periodontal tissues of patients with periodontitis and DM. AGEs aggravate periodontitis with DM by increasing the expression of inflammation-related factors in periodontal tissues. 6-Shogaol, a major compound in ginger, has anti-inflammatory and anti-oxidative activities. However, the influence of shogaol on DM-associated periodontitis is not well known. In this study, the effects of 6-shogaol on AGEs-induced oxidative and anti-oxidative responses, and IL-6 and ICAM-1 expression in human gingival fibroblasts (HGFs) were investigated. When HGFs were cultured with 6-shogaol and AGEs, the activities of reactive oxygen species (ROS) and antioxidant enzymes (heme oxygenase-1 [HO-1] and NAD(P)H quinone dehydrogenase 1 [NQO1]), and IL-6 and ICAM-1 expressions were investigated. RAGE expression and phosphorylation of MAPKs and NF-κB were examined by western blotting. 6-Shogaol significantly inhibited AGEs-induced ROS activity, and increased HO-1 and NQO1 levels compared with the AGEs-treated cells. The AGEs-stimulated expression levels of receptor of AGE (RAGE), IL-6 and ICAM-1 and the phosphorylation of p38, ERK and p65 were attenuated by 6-shogaol. These results suggested that 6-shogaol inhibits AGEs-induced inflammatory responses by regulating oxidative and anti-oxidative activities and may have protective effects on periodontitis with DM

High Glucose Induces Severe Periodontitis.

Background/Aims: Diabetic patients are susceptible to severe periodontitis, but the precise mechanism is not fully understood. Aim of this study was to explore the biological pathogenesis of severe periodontitis in diabetic patients focusing on the crosstalk of human gingival fibroblasts (HGFs) and macrophages. Methods: A total of 70 periodontitis patients with or without diabetes mellitus (DM) were enrolled, and the statistical relationships of diabetic conditions to the periodontal inflammatory parameters were examined by cross-sectional study. In in vitro study, HGFs cell line CRL-2014® (ATCC) and differentiated THP-1 macrophages were cultured with normal glucose (NG: 5.5 mM) or high glucose (HG: 25 mM) condition, and treated with indicated inflammatory factors such as calprotectin (CPT), interleukin (IL)-1β and IL-6. To examine the effects of HG on soluble IL-6 receptor (sIL-6R) production in THP-1 macrophages, the supernatants were collected and the sIL-6R levels were measured by ELISA. To examine the effects of HG on IL-1β or IL-6-induced matrix metalloproteinase (MMPs) production in HGFs, the supernatants were collected. Levels of MMP-1 and tissue inhibitor of MMP-1 (TIMP-1) were measured by ELISA. Finally, after conditioned medium (CM) from THP-1 macrophages cultured with NG or HG conditions was collected, HGFs were treated with the CM. The supernatants were collected 24 hours later and the levels of MMP-1 and TIMP-1 were measured. To examine the specific effects of IL-1β contained in CM on MMP-1 and TIMP-1 production in HGFs, IL-1 receptor antagonist (IL-1ra) was used. Results: There were statistical correlation between IL-1β and sIL-6R levels in gingival crevicular fluid (GCF) and HbA1c in periodontitis patients with DM (IL-1β: P=0.035, sIL-6R: P=0.040). HG and CPT significantly induced sIL-6R production in THP-1 macrophages. HG significantly enhanced IL-1β or IL-6/sIL-6R-induced MMP-1 production in HGFs. The increase of MMP-1 by both IL-1β and IL-6/sIL-6R was significantly inhibited by specific ERK or IκB inhibitors. Corresponding to the regulation of MMP-1 production, HG condition increased the phosphorylation of p44/42 MAPK and IκBα in HGFs treated with IL-1β or IL-6/sIL-6R. Finally, MMP-1 production in HGFs cultured with HG increased significantly by CM from THP-1 macrophages cultured with HG. The induction of MMP-1 by the CM from THP-1 macrophages cultured with HG was significantly inhibited by dose dependent of IL-1ra in HGFs cultured with HG. Conclusion: Diabetic conditions such as HG induce IL-1β and sIL-6R production from macrophages in inflammatory periodontal tissues and may exacerbate the periodontitis synergistically via MMP-1 production from HGFs

National survey of the association of depressive symptoms with the number of off duty and on-call, and sleep hours among physicians working in Japanese hospitals: a cross sectional study

<p>Abstract</p> <p>Background</p> <p>Physicians' mental health may be adversely affected by the number of days of work and time spent on-call, and improved by sleep and days-off. The aim of this study was to determine the associations of depressive symptoms with taking days of off duty, hours of sleep, and the number of days of on-call and overnight work among physicians working in Japanese hospitals.</p> <p>Methods</p> <p>A cross-sectional study as a national survey was conducted by mail. The study population was 10,000 randomly selected physicians working in hospitals who were also members of the Japan Medical Association (response rate 40.5%). Self-reported anonymous questionnaire was sent to assess the number of days off-duty, overnight work, and on-calls, and the average number of sleep hours on days not working overnight in the previous one month. Depressive state was determined by the Japanese version of the Quick Inventory of Depressive Symptomatology. Logistic regression analysis was used to explore the associations between depressive symptoms and the studied variables.</p> <p>Results</p> <p>Among the respondents, 8.3% of men and 10.5% of women were determined to be depressed. For both men and women, depressive state was associated with having no off-duty days and averaging less than 5 hours of sleep on days not doing overnight work. Depressive state was positively associated with being on-call more than 5 days per month for men, and more than 8 days per month for women, and was negatively associated with being off-duty more than 8 days per month for men.</p> <p>Conclusion</p> <p>Some physicians need some support to maintain their mental health. Physicians who do not take enough days-off, who reduced sleep hours, and who have certain number of days on-calls may develop depressive symptoms.</p

Níveis séricos de sódio e potássio em pacientes com Málaria causada pelo Plasmodium vivax em uma unidade de referência para diagnóstico e tratamento da doença

A malária continua sendo, uma das principais doenças infecciosas em todo o mundo. O Plasmodium vivax é a principal espécie causadora de malária no Brasil. O objetivo do presente estudo foi investigar os níveis séricos dos eletrólitos sódio e potássio em pacientes com infecção pelo Plasmodium vivax, bem como sua relação com parâmetros clínicos e laboratoriais de potencial gravidade da doença. Trata-se de um estudo epidemiológico descritivo, sendo avaliadas as concentrações séricas dos eletrólitos sódio e potássio. Foram incluídos 153 pacientes de junho de 2010 a junho de 2016. Neste estudo, a redução dos níveis da concentração média de sódio foi observada no grupo de pacientes que apresentava febre no dia da consulta e temperatura axilar acima de 37.5 °C.No entanto, o mesmo não foi observado em relação ao potássio. Dessa forma, podemos concluir que na malária vivax a concentração sérica de sódio é menor nos pacientes que relatam ter tido febre no dia da consulta e naqueles que apresentam temperatura axilar maior que 37.5 °C. Os níveis séricos de potássio não são diferentes entre os pacientes que apresentaram febre. Sendo assim, o presente estudo ressalta a necessidade do monitoramento dos níveis de sódio nos pacientes com malária aguda causada pelo Plasmodium vivax